1. Field of the Invention
This invention relates to a process by which ozone oxidizable compounds are separated from non-oxidizable compounds in biomass extracts. More particularly, this invention relates to the separation of cephalomannine and related ozone oxidizable compounds from taxol and other non-oxidizable taxaries in extracts of biomass containing a complex mixture of compounds.
2. Description of the State of Art
Between the years 1958 and 1980, extracts of over 35,000 plant species were tested for anticancer activity as part of an NCI-sponsored program. Chemists Monroe E. Wall and M.C. Wani first isolated a crude extract concentrate from yew tree (Taxus brevifolia) bark and wood samples in 1963. Initial screening showed the extract to be a potential anticancer agent, being very active against an unusually wide range of rodent cancers. Isolation of the active agent in the crude extract took several years due to the very low concentrations of the agent present in the plants and due to the difficulty of separating the closely-related diterpenoids. The active agent was identified, the structure determined and the compound was named taxol, in 1971. Despite taxol's excellent activity in model tumor systems, clinical trials were delayed owing to short supplies of the drug and formulation problems related to the drug's low solubility.
In ongoing clinical trials, taxol has shown promising results in fighting advanced cases of ovarian, breast and other cancers. Recently, taxol was approved by the Food and Drug Administration for the treatment of refractory ovarian cancer; however, taxol is extracted in limited quantities from a natural vegetation that is in short supply.
Currently, taxol is isolated on a large scale only from the bark of Taxus brevifolia. However, taxol can be potentially isolated on a large commercial scale from other parts of Taxus and also from cell cultures of Taxus. See U.S. Pat. No. 5,019,504, issued to Christen et al. The method of isolation of taxol from any natural source is complex. A particularly difficult and expensive part of the isolation of taxol is the separation of the closely-related diterpenoid cephalomannine. The only structural difference between taxol and cephalomannine, as shown in structures 1 and 2 of FIG. 2, respectively, involves the side chain portion of the compound, thus giving rise to similar chemical properties. Achieving a clean separation of these closely related compounds is extremely difficult, but can be accomplished by various means of chromatography. However, the use of chromatography on a commercial scale is an extremely expensive step in the process of separating compounds and would be ultimately reflected in the cost of the drug.
There have been some methods developed for improving the separation of taxol from cephalomannine; however, chromatography is still required for a complete separation of the two compounds. For example, Kingston et al. reported a method, under stoichiometric or catalytic conditions, to modify the side chain of cephalomannine in the presence of taxol in purified and/or partially-purified taxane mixtures. See, Kingston et al. Journal of Organic Chemistry, 55 (2): 259-261 (1992), incorporated herein by reference. In the partially-purified mixture, disclosed by Kingston et al., taxol and cephalomannine were present in a total amount of about 80% by weight. Selective oxidation of the tiglate group of cephalomannine using osmium tetroxide under stoichiometric or catalytic conditions yielded a diol, shown in FIG. 3 as structure 3, and did not affect the structure of taxol. The diol was separated from the reaction mixture by silica gel chromatography as shown in FIGS. 3 and 4.
The above technical paper by Kingston et al. discloses a process whereby the chemical properties of cephalomannine are altered thus allowing for a cleaner separation of taxol from the newly formed diol; however, as explained in further detail below, the Kingston et al. process is not suitable for use with unpurified taxane mixtures. A very long reaction time is required for the complete oxidation of cephalomannine by osmium tetroxide. Furthermore, the very high ratio of osmium tetroxide to cephalomannine results in a two-fold disadvantage. First, the reagent osmium tetroxide is very expensive, i.e., $76.00 per gram from one common commercial supplier in 1993. Second, due to the extreme toxicity of osmium tetroxide, careful handling is required for both the reagent and the resulting waste associated with the process.
There is still a need, therefore, for an inexpensive, simple, safe and effective separation of cephalomannine from taxol at a wide range of purities.